We have continued to develop our biophysical measurement systems (biological atomic force microscopy (Bio-AFM) platforms, Quartz Crystal Microbalance-Dissipation (QCM-D), and optical microscopy and spectroscopy), and to apply these technologies to important biomedical investigations in collaboration with outstanding NIH intramural and extramural scientists. On the instrumentation front, we have enhanced a new high-speed AFM from home-made and open-source components such us a DVD pickup head and NI Labview for scan rates up to twenty lines per second and performed in-fluid imaging. We have received a significant collection of AFM and nanotechnology instruments from NIAID's Dr. Thomas Wellems and Dr. Fuyuki Tokumasu (now at University of Tokyo) for ongoing upgrades and integration with advanced optical imaging. We are working toward broader and more insightful applications of multifunctional, multimodal, and multiplatform AFM imaging and single molecule force spectroscopy (SMFS) for cellular and macromolecular sample characterizations. On biomedical applications in this year, we have continued our broad range of collaborations that include: (1) We have advanced our commitment to developing a better clinical vaccine toward enhanced immunological response and eventual eradication of malaria. We have investigated the macromolecular structure and nanomechanical properties of more malaria vaccine candidates and virus-like-particle (VLP) or liposome carriers via Bio-AFM and related bioanalysis with Dr. David Narum (NIAID, NIH) and more collaborators. These malaria protein antigens and vaccine carriers are produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up production. Biophysical characterization at single macromolecule and assembly level using Bio-AFM imaging and force spectroscopy are helping define these vaccine constructs along the developmental phases. We also aim to improve mechanistic understanding of the malaria parasites and pathogen-host interactions, especially toward vaccine considerations. We have contributed to the published observation of a reversible conformation change and adhesion domain masking in the Plasmodium falciparum circumsporozoite protein (CSP), which is the leading malaria vaccine target. (2) We have expanded our collaboration on multifunctional nanomedicine and theranostics with Dr. Xiaoyuan Chen (laboratory of Molecular Imaging and Nanomedicine, NIBIB) and a large international team of co-investigators including Dr. Peng Huang (NIBIB) and Zhe Wang (NIBIB). We have contributed to several published studies such as using polymeric nanovehicles to regulate the differentiation dynamics of transplanted neural stem cells after traumatic brain injury and using tumor-specific formation of enzyme-instructed supramolecular assemblies as cancer theranostics. We are examining critically broader applications of Bio-AFM and QCM-D methodology for investigating nanoparticle theranostics, characteristics of the cancer cells and stem cells, and related biomedical systems. (3) We have collaborated with Dr. Andrew Doyle and Dr. Kenneth Yamada (Laboratory of Cell and Developmental Biology, NIDCR) on nanomechanics and structural properties of reconstituted extra cellular matrix (ECM)-like collagen gels via Bio-AFM force spectroscopy to investigate cell adhesion, migration, and other dynamic behaviors. A manuscript entitled Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions is under review. Collaborating also with Dr. Raimon Sunyer (Institute for Bioengineering of Catalonia, Spain), we are defining force spectroscopy approaches to explore more completely tissue-mimicking soft material and tissue specific extracellular matrices. (4) We have continued our Bio-AFM studies of protein clathrin and assemblies with collaborators including Prof. Eileen Lafer and Prof. Rui Sousa (Univ. Texas Health Sciences Center, San Antonio), Dr. Ralph Nossal (NICHD) and Dr. Dan Sackett (NICHD). Clathrin is a key protein for receptor-mediated endocytosis and intracellular trafficking. Further Bio-AFM and QCM-D measurements have been pursued this year to characterize clathrin and its assembled structures, as well as interaction with several partner proteins important to the function of cells. This collaboration also covers bio-AFM studies of microtubules and their interactions with certain cancer drugs. In this area of cellular exocytosis and endocytosis, we have also continued to investigate with Dr. Ling-gang Wu (NINDS) and coworkers cell membrane and intracellular structural changes with Dr. Ling-gang Wu (NINDS) and coworkers to better understand synaptic transmission in brain. A manuscript is under review. (5) Among other continuing and new collaborations this year, we have investigated protein and DNA interactions and nucleosome structure and dynamics, critically important in gene regulation and disease mechanisms, with Dr. Yawen Bai (Laboratory of Molecular Cell Biology, NCI) and coworkers. With Dr. Richard Hendler(NHLBI) and others, we have advanced our Bio-AFM and biophysical studies of several protein fiber assemblies, such as amyloid-beta fibrils in the Alzheimer's disease, to investigate time-resolved assembly pathways in physiologically relevant fluid environments. We have collaborated with Prof. Xiangyun Qiu (George Washington University) and his group on DNA cross-linking and related studies. We have started to work with Dr. Yun-Xing Wang (NCI) on determining high-resolution structures of a key human immunodeficiency virus (HIV) RNA and its complex with co-factors. We have collaborated on projects with Prof. Qi Lu (Delaware State University), Prof. Hacene Boukari and others on the effects of nanoparticles on the organization of lipid domains toward cancer treatment. Finally, we have initiated a new collaboration this year focused on adaptive and collective behaviors in cancer cells, with Prof. Wolfgang Losert (University of Maryland at College Park), Dr. David Rampulla (Plexus Institute) and others, contributing on combined AFM force spectroscopy and optical microscopy observations.